Molecular Mechanism of Drug-Dependent Ribosome Stalling

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162

179

Significance Macrolide antibiotics inhibit translation by binding in the ribosomal nascent peptide exit tunnel. It was believed that macrolides interfere with protein synthesis by obstructing the egress of nascent proteins. In contrast to this view, the results of ribosome profiling analysis suggest that the main mode of macrolide action is context-specific inhibition of peptide bond formation. The ribosome with a macrolide molecule bound in the tunnel is impaired in catalysis of peptide bond formation between specific combinations of the peptidyl donors and aminoacyl acceptors, leading to interruption of translation when such problematic substrates are encountered. These findings underscore the existence of a link between the ribosomal tunnel and the peptidyl transferase center and pave the way for development of superior antibiotics.

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 90%

The general mode of translation inhibition by macrolide antibiotics

Kannan

Kanabar

Schryer

et al. 2014

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162

179

“…3C). Similarly, minute modifications in the structure of the antibiotics or in their binding sites may have significant effects on their binding and properties (20,37,54,55). LM on its own is a less potent protein synthesis inhibitor than ERY.…”

Section: Discussionmentioning

confidence: 99%

The structure of ribosome-lankacidin complex reveals ribosomal sites for synergistic antibiotics

Auerbach

Mermershtain

Davidovich

et al. 2010

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Crystallographic analysis revealed that the 17-member polyketide antibiotic lankacidin produced by Streptomyces rochei binds at the peptidyl transferase center of the eubacterial large ribosomal subunit. Biochemical and functional studies verified this finding and showed interference with peptide bond formation. Chemical probing indicated that the macrolide lankamycin, a second antibiotic produced by the same species, binds at a neighboring site, at the ribosome exit tunnel. These two antibiotics can bind to the ribosome simultaneously and display synergy in inhibiting bacterial growth. The binding site of lankacidin and lankamycin partially overlap with the binding site of another pair of synergistic antibiotics, the streptogramins. Thus, at least two pairs of structurally dissimilar compounds have been selected in the course of evolution to act synergistically by targeting neighboring sites in the ribosome. These results underscore the importance of the corresponding ribosomal sites for development of clinically relevant synergistic antibiotics and demonstrate the utility of structural analysis for providing new directions for drug discovery.lankamycin | ribosomes | synergism | resistance | rRNA

“…Although either of these mechanisms are feasible, we favor the second possibility, which is consistent with our findings that blocking efflux with PA␤N makes cells more sensitive to erythromycin and deleting either the AcrA/AcrB or TolC components of the AcrAB-TolC efflux pump reduces the erythromycin resistance of a strain with ⌬MKR-L22-titin ribosomes compared with a strain with L22-titin ribosomes. The ⌬MKR mutation is known to affect translation of certain mRNAs by reducing programmed ribosome stalling (36,37). How might ⌬MKR-linked translational changes affect macrolide uptake or efflux?…”

Section: Discussionmentioning

confidence: 99%

Revisiting the mechanism of macrolide-antibiotic resistance mediated by ribosomal protein L22

Moore

Sauer²

2008

Bacterial antibiotic resistance can occur by many mechanisms. An intriguing class of mutants is resistant to macrolide antibiotics even though these drugs still bind to their targets. For example, a 3-residue deletion (⌬MKR) in ribosomal protein L22 distorts a loop that forms a constriction in the ribosome exit tunnel, apparently allowing nascent-chain egress and translation in the presence of bound macrolides. Here, however, we demonstrate that ⌬MKR and wild-type ribosomes show comparable macrolide sensitivity in vitro. In Escherichia coli, we find that this mutation reduces antibiotic occupancy of the target site on ribosomes in a manner largely dependent on the AcrAB-TolC efflux system. We propose a model for antibiotic resistance in which ⌬MKR ribosomes alter the translation of specific proteins, possibly via changes in programmed stalling, and modify the cell envelope in a manner that lowers steady-state macrolide levels.ermC ͉ erythromycin ͉ ribosome ͉ TolC M any antibiotics inhibit bacterial protein synthesis. Understanding how microbes become antibiotic resistant is important both for developing effective treatment regimens and designing new therapeutics. Macrolides consist of a 14-to 16-member lactone ring with different appended sugars and comprise a key group of inhibitors of bacterial translation (1, 2). The inhibitory activity of macrolides, including erythromycin, depends on binding to a site near the polypeptide exit tunnel of the large ribosomal subunit (3, 4). Because macrolides do not bind to ribosomes with an occupied exit tunnel and cause the synthesis of 2-10 residue peptides in translation assays in vitro, it has been proposed that drug binding physically blocks elongation of nascent proteins beyond this size (5-7).Some macrolide-resistance mutations alter the ribosomal target site and prevent binding (4,8). Intriguingly, other mutations confer resistance despite the fact that macrolides still bind the mutant ribosome well (8-10). For example, deletion of the M 82 K 83 R 84 sequence in Escherichia coli ribosomal protein L22 (⌬MKR) allows growth in the presence of high levels of erythromycin and other macrolides (11-13). The same mutation makes Haemophilus influenzae resistant to numerous macrolides (14); different L22 mutations also confer macrolide resistance in other bacterial species (2). When binding has been measured, ribosomes with macrolideresistant alterations in L22 bind erythromycin with near wild-type affinity (8,10,12,15).In a crystal structure of the E. coli ribosome, the MKR sequence is part of an extended L22 loop, which together with a similar loop in protein L4 forms a narrow constriction in the exit tunnel (Fig. 1A) (16, 17). Cryo-EM studies initially revealed a widened exit tunnel in E. coli ⌬MKR ribosomes (18). This loop is also displaced to create an expanded tunnel in structures of ⌬MKR ribosomes from Thermus thermophilus and Haloarcula marismortui (4, 19). These results explain the altered chemical reactivity in E. coli ⌬MKR ribosomes of 23S-RNA bases (13). Together,...